Creating artificial microvessel networks that structurally and functionally mimic the native microvasculature is critical for the fabrication and survival of a wide range of bioengineered tissues, and will also provide realistic cost-effective i vitro models for drug discovery, product testing and toxicology screening. We have recently developed an innovative, non-invasive ultrasound-based method to spatially pattern cells within 3D hydrogels, and have demonstrated the feasibility of translating ultrasound technologies to microvascular tissue engineering. We have shown that acoustic radiation forces associated with ultrasound standing wave fields (USWF) can rapidly organize cells into distinct multicellular planar bands within 3D collagen gels. USWF-induced patterning of endothelial cells rapidly initiates the assembly of complex, branching vessel networks throughout the volume of the hydrogel. Importantly, the rate of formation as well as the morphology of resultant microvascular networks can be controlled by design of the acoustic field. The goal of this project is to advance the use of USWF technologies as a versatile, non-invasive method to vascularize hydrogels in vitro, with the ultimate goal of translating this technique to in situ vascularization. To do so, w will (1) identify USWF exposure parameters that stimulate microvessel assembly and control microvessel structure, (2) identify critical biological parameters that influence the structure and function of USWF-fabricated microvascular networks, and optimize their usage for in vitro and in situ fabrication, and (3) assess the in vivo performance of USWF hydrogel constructs fabricated in vitro and in situ. Non-invasive ultrasound-based technologies that can rapidly organize cells into distinct geometric patterns, either in vitro or in situ, will be broadly applicable across cel and tissue types, and thus, are expected to have a wide impact on advancing tissue engineering and regenerative medicine.